αha clone 3f10

Immunofluorescence of endogenous levels of Cln3-3HA was carried out by a signal amplification method (Vergés et al, 2007 (link)) with αHA (clone 3F10; Roche) and goat-αrat and rabbit-αgoat Alexa555-labeled antibodies (Molecular Probes) on methanol-pemeabilized cells. To analyze localization of Cln3-3HA, we used N2CJ, a plugin for ImageJ (Wayne Rasband, NIH), to perform accurate quantification in both cytoplasmic and nuclear compartments of cells (Yahya et al, 2014 (link)).

The plasmids pMT/V5His and pAc5/V5His (Invitrogen) were used as backbones for tag insertions and removals, and for subcloning of the constructs pMT/FlagHis, pMT/HAFlag, pAc5/Diap2, pMT/Dredd-V5His, pMT/Dredd-HAFlag, pMT/PGRP-LCx-Myc, pMT/Kenny-HA, pMT/Drice and pMT/ALG(p20/p10)Drice-V5Flag. The point mutation Drice^C211A was made by site-directed mutagenesis (Agilent Technologies, Santa Clara, California, USA or Stratagene, San Diego, California, USA). GST-TUBE (tandem ubiquitin entity) was provided by Dr. Mads Gyrd-Hansen. The following antibodies were used: α-K63 (clone Apu3, #05-1308, Millipore, Burlington, Massachusetts, USA), α-Diap2 [33 (link)], α-Drice [34 (link)], α-HA (clone 3F10, #11867423001, Roche, Basel, Switzerland), α-V5 (Clone SV5-Pk1, #MCA1360, Bio-Rad, Hercules, California, USA), α-Myc (#M4439, Sigma), α-phosphohistone H3 (PHH-3) (Ser10, #9701, Cell Signalling Technology, Danvers, Massachusetts, USA) and α-Actin (C-11, sc-1615, Santa Cruz Biotechnology, Dallas, Texas, USA).